Infection of Human Immunodeficiency Virus and other retroviruses results in the stable incorporation of the viral genome into the host DNA. Inhibition of early events of an infectious cycle have the potential to block the permanent residency of the virus in the cell. This research focuses on the replication of the viral RNA into double-stranded (ds) DNA catalyzed by the viral reverse transcriptase (RT), an early event in the infectious pathway. Replication of the virus requires coordination between the polymerase and RNase H activities of RT. A key area of investigation is the RNase H activity, which is essential at multiple stages of viral replication. Research defining the mechanism involved in the recognition of RNA/DNA hybrids during polymerase dependent and independent RNase H cleavages are proposed. The HIV-1 RNase H shows defined specificity which distinguish it from cellular enzymes. This proposal studies the unique cleavage associated with the removal of the tRNA primer and the subsequent requirements for plus-strand transfer. The sequence and/or structural features of this recognition will be examined through comparative studies of alternative tRNAs. The requirement for entry of the acceptor molecule will be defined. Assays have been developed for plus-strand transfer reaction. These assays will be extended towards the development of an in vitro replication system. Biochemical analysis of RT/RNase H will included cross-linking studies to identify specific residues in contact with RNA/DNA hybrids, with a particular focus on the unique tRNA/DNA substrate mimics. Mutagenesis studies of targeted amino acids will define requirements for enzyme multimerization, substrate binding, and directional processing.